Lm Tissue Preparation

BIO 513 ANIMAL TISSUE PREPARATION – LIGHT MICROSCOPY

 RINSE IN COLD 0.1M PHOSPHATE BUFFER SOLUTION

 GLUTARALDEHYDE FIXATIVE ...... overnight - 24 hours  0.1 M PHOSPHATE BUFFER ...... 3 x 10 min. changes  70% ETHYL ALCOHOL ...... 1 hour ** Tissue can be stored in this solution**  95% ETHYL ALCOHOL ...... 1 hour  ABSOLUTE ETHYL ALCOHOL ...... 1 hour ** All water must be removed from tissue at this step **  ABSOLUTE ETHYL ALCOHOL - HISTOCLEAR . . . . . 1 hour

 HISTOCLEAR I ...... 1 hour

 HISTOCLEAR II ...... 1 hour Transfer vials to vacuum oven (T = 60 °C).  MELTED PARAFFIN I ...... 30 minutes

 MELTED PARAFFIN II ...... 30 minutes

 MELTED PARAFFIN III ...... 30 minutes

 EMBED IN FRESH PARAFFIN Use embedding station to embed tissue. Turn on heating unit at least 2 hours before use. Paraffin blocks can be stored indefinitely if kept from heat and dust. Store blocks in the freezer inside cardboard containers. Whilst sectioning keep blocks cool with iced water.